Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

A pharmaceutical composition for treating or relieving inflammatory bowel
disease (IBD) is provided. The pharmaceutical composition includes an
extract of Bupleurum as an active ingredient, in which the Bupleurum is
selected from the group consisting of Bupleurum krylovianum, Bupleurum
chinense, Bupleurum commelynoideum, Bupleurum scorzonerifolium, Bupleurum
triradiatum, Bupleurum falcatum, Bupleurum kaoi and a combination
thereof.

Claims:

1. A pharmaceutical composition for treating or relieving inflammatory
bowel disease, comprising an extract of Bupleurum as an active
ingredient.

2. The pharmaceutical composition as claimed in claim 1, wherein the
Bupleurum is selected from a species which is slightly cold in nature and
bitter to the taste.

4. The pharmaceutical composition as claimed in claim 1, wherein the
extract of Bupleurum is extracted from the root thereof.

5. The pharmaceutical composition as claimed in claim 1, wherein the
Bupleurum is extracted with an ethanol aqueous solution in a
concentration of about 20.about.95% by volume.

6. The pharmaceutical composition as claimed in claim 5, wherein the
ethanol aqueous solution is in a concentration of about 40.about.95% by
volume.

7. The pharmaceutical composition as claimed in claim 1, wherein the
inflammatory bowel disease comprises Crohn's disease, enteritis or a
bowel disease with an inflammatory symptom.

8. A method for treating or relieving inflammatory bowel disease,
comprising administrating an extract of Bupleurum to a subject.

9. The method as claimed in claim 8, wherein the Bupleurum is selected
from a species which is slightly cold in nature and bitter to the taste

10. The method as claimed in claim 8, wherein the Bupleurum is selected
from the group consisting of Bupleurum krylovianum, Bupleurum chinense,
Bupleurum commelynoideum, Bupleurum scorzonerifolium, Bupleurum
triradiatum, Bupleurum falcatum, Bupleurum kaoi and a combination
thereof.

11. The method as claimed in claim 8, wherein the extract of Bupleurum is
extracted from the root thereof.

12. The method as claimed in claim 8, wherein the Bupleurum is extracted
with an ethanol aqueous solution in a concentration of about 20.about.95%
by volume.

13. The method as claimed in claim 12, wherein the ethanol aqueous
solution is in a concentration of about 40.about.95% by volume.

14. The method as claimed in claim 8, wherein the inflammatory bowel
disease comprises Crohn's disease, enteritis or a bowel disease with an
inflammatory symptom.

15. The method as claimed in claim 8, wherein the extract of Bupleurum is
orally administrated.

16. The method as claimed in claim 8, wherein the subject comprises a
human.

17. A method for preparing a medicament of treating or relieving
inflammatory bowel disease, comprising: providing an extract of Bupleurum
as an active ingredient, in preparation of a medicament of treating or
relieving inflammatory bowel disease.

18. The method as claimed in claim 17, wherein the Bupleurum is selected
from a species which is slightly cold in nature and bitter to the taste.

19. The method as claimed in claim 17, wherein the Bupleurum is selected
from the group consisting of Bupleurum krylovianum, Bupleurum chinense,
Bupleurum commelynoideum, Bupleurum scorzonerifolium, Bupleurum
triradiatum, Bupleurum falcatum, Bupleurum kaoi and a combination
thereof.

20. The method as claimed in claim 17, wherein the extract of Bupleurum
is extracted from the root thereof.

21. The method as claimed in claim 17, wherein the Bupleurum is extracted
with an ethanol aqueous solution in a concentration of about 20.about.95%
by volume.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application
No. 61/360,190, filed Jun. 30, 2010, and claims the priority of Taiwan
Patent Application No. 099143928, filed Dec. 15, 2010, the entire
contents of which are hereby incorporated by reference.

BACKGROUND

[0002] 1. Technical Field

[0003] The disclosure relates to a pharmaceutical composition for treating
or relieving inflammatory bowel disease (IBD), and in particular relates
to an extract of Bupleurum as an active ingredient for treating or
relieving inflammatory bowel disease (IBD).

[0004] 2. Description of the Related Art

[0005] The current medicament for inflammatory bowel disease (IBD) is
commonly categorized into four groups, (1) immunosuppression drugs, such
as azathioprine, cyclosporine or steroids, (2) therapeutic agents, such
as sulfsalazine or 5-aminosalicylic acid (5-ASA), (3) biological agents
for inhibiting specific inflammatory factors, such as infliximab or
remicade, and (4) anti-diarrheal agents. However, these medicaments may
not completely cure IBD and sometimes cause side effects.

[0006] A new generation of drugs for rheumatoid arthritis by inhibiting
the tumor necrosis factor (TNF-α) has also been used for the
treatment of IBD, such as etanercept, adalimumab, or the like. These
drugs show good drug effects during a short period of time and immune
tolerance, but due to its price, intravenous injection, a potential
immunoresponse and limited test data, clinical use has been limited.

[0007] Bupleurum is a medicinal plant recorded in a Chinese herbal
medicine book, Shennong bencao jiing. It is traditionally used for
relieving fever, pain and inflammatory diseases and treating irritations,
infections causing fever, thirst, jaundice and hepatitis, etc. The
Bupleurum root is known for its medicinal effect and has been chemically
isolated. Saikosaponin, longispinogenin, steroids, fatty oils, flavonoids
and saccharides have been isolated from the extract of the Bupleurum
root, in which saikosaponin is the primary component.

[0008] It is known that attempts have been made in biological medicine, to
use Bupleurum in treating hepatitis. Nevertheless, no study or reference
for using Bupleurum to treat IBD has been found. Accordingly, a specific
extraction method of Bupleurum was used to develop a novel pharmaceutical
composition and method for treating or relieving IBD, wherein the
correlations between a Bupleurum extract and IBD were identified.

SUMMARY

[0009] A detailed description is given in the following embodiments with
reference to the accompanying drawings.

[0010] In one embodiment of the disclosure, a pharmaceutical composition
for treating or relieving inflammatory bowel disease is provided, which
comprises an extract of Bupleurum as an active ingredient.

[0011] In another embodiment of the disclosure, a method for treating or
relieving inflammatory bowel disease is provided, which comprises
administrating an extract of Bupleurum to a subject. The subject includes
a human.

[0012] In a further embodiment of the disclosure, a method for preparing a
medicament of treating or relieving inflammatory bowel disease is
provided, which comprises a step of providing an extract of Bupleurum as
an active ingredient in preparation of a medicament of treating or
relieving inflammatory bowel disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The disclosure can be more fully understood by reading the
subsequent detailed description and examples with references made to the
accompanying drawings, wherein:

[0014] FIG. 1 is the amount of TNF-α in a bowel infusion, in which
column (A) refers to a negative control, column (B) refers to a vehicle
treatment with an enteritis induction of TNBS and no administration of a
Bupleurum extract, column (C) refers to one embodiment of the invention
with oral administration of 100 mg/kg of a Bupleurum krylovianum extract
by the enteritis animal model, and column (D) refers to a positive
control with oral administration of 200 mg/kg of 5-ASA by the enteritis
animal model;

[0015]FIG. 2 is the amount of IL-6 in a bowel infusion, in which the
representation of the columns are identical to those in FIG. 1;

[0016]FIG. 3 is the amount of G-CSF in a bowel infusion, in which the
representation of the columns are identical to those in FIG. 1;

[0017]FIG. 4 is the amount of IL1-β in a bowel infusion, in which
the representation of the columns are identical to those in FIG. 1;

[0018]FIG. 5 is the type of the bowel in the negative control with an
injection of a 50% ethanol aqueous solution without TNBS, in which the
diameter of the bowel is about 1 mm and the appearance of the feces is
semi-solid;

[0019] FIG. 6 is the type of the bowel in the vehicle treatment with an
enteritis induction of TNBS and no administration of a Bupleurum extract,
in which the diameter of the bowel is about 3˜5 mm and the
appearance of the feces is mushy and watery;

[0020] FIG. 7 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum krylovianum extract (100 mg/kg DLS01=080221-001);

[0021] FIG. 8 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 1000 mg/kg
of a Bupleurum krylovianum extract (1000 mg/kg DLS01=080221-001);

[0022] FIG. 9 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum chinense extract (DLS01-12 100 mg/kg);

[0023] FIG. 10 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum commelynoideum extract (DLS01-41 100 mg/kg);

[0024] FIG. 11 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum scorzonerifolium extract (DLS01-53 100 mg/kg);

[0025] FIG. 12 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum triradiatum extract (DLS01-69 100 mg/kg);

[0026] FIG. 13 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum falcatum extract (BEL-0506 100 mg/lg);

[0027] FIG. 14 is the type of the bowel in one embodiment of the invention
with an enteritis induction of TNBS and oral administration of 100 mg/kg
of a Bupleurum kaoi extract (BEL-1153 100 mg/lg); and

[0028]FIG. 15 is the type of the bowel in the positive control with an
enteritis induction of TNBS and oral administration of 200 mg/kg of 5-ASA
(200 mg/kg 5-ASAl).

DETAILED DESCRIPTION

[0029] The following description is of the best-contemplated mode of
carrying out the disclosure. This description is made for the purpose of
illustrating the general principles of the invention and should not be
taken in a limiting sense. The scope of the invention is best determined
by reference to the appended claims.

[0030] The Bupleurum species in the disclosure are selected from the
family which is slightly cold in nature and bitter to the taste according
to page-78 in "The English-Chinese Encyclopedia of Practical Traditional
Chinese medicine vol. 2: The Chinese Materia Medica" (Higher Education
Press, Beijing, China, 1994 edition 1.)

[0031] More specific, the Bupleurum species in the application are
selected from the group consisting of Bupleurum krylovianum, Bupleurum
chinense, Bupleurum commelynoideum, Bupleurum scorzonerifolium, Bupleurum
triradiatum, Bupleurum falcatum, Bupleurum kaoi and a combination
thereof.

[0032] The Bupleurum can be extracted from the root of a plant or a whole
plant as described in U.S. patent application Ser. No. 12/829,357 (Jul.
1, 2010) which is incorporated by reference. The "extraction" according
to the disclosure can be solvent extraction. The "solvent extraction" is
directed to a method comprising: adding a substrate of interest into a
suitable solvent and extracting a target compound based on the solubility
of components of the substrate in the solvent. In one example, the
Bupleurum is ground and immerged in a solvent under room temperature for
a period of time. The solvent is isolated and dried under room
temperature to obtain an extract of the Bupleurum. In another example,
the ground Bupleurum is added into a polar solvent and extracted after
heating under reflux.

[0033] The solvent used in the invention can be C1˜C12
alcohols, C2˜C5 acetates, C5˜C6 alkanes,
or combinations thereof, such as methanol, ethanol, propanol,
isopropanol, butanol, 2-butyl alcohol, tert-butyl alcohol, 1,3-butandiol,
1,4butandiol, pentanol, isopentanol, 2,3-pentandiol, 2,4-pentandiol,
cyclopentanol, hexanol, cyclohexanol, heptanol, octanol, nonanol,
decanol, undecanol, dodecanol, ethyl acetate, propyl acetate, pentyl
acetate, pentane, cyclopentane, hexane, cyclohexane, or combinations
thereof, but are not limited thereto. In one example, ethanol, ethyl
acetate and/or pentane is/are used as a solvent for extraction. In
another example, an ethanol aqueous solution is used as a solvent for
extraction. The concentration of ethanol can be about 20%˜95%, or
about 40%˜95% based on the ethanol aqueous solution.

[0034] The volume of the solvent can be more than 5 times that of the
Bupleurum, or about 5˜10 times.

[0035] The extraction may last more than two hours. In one embodiment, the
extraction lasts for about 2˜24 hours or about for 4˜5 hours.

[0036] The extraction is usually performed at room temperature. In one
embodiment, the extraction is performed at a temperature from room
temperature to the temperature of the ethanol aqueous solution is boiled
and refluxed.

[0037] The extraction may further comprise a step of concentration by
drying, wherein a solid or crystal form is extracted after being heated
and refluxed. The extraction process can be repeated several times for a
pure extract.

[0038] The animal model of enteritis for test is from an induction by
trinitrobenzene sulfonic acid (TNBS) (Current Protocols in Immunology
(2001) 15.19.1-15.19.14; Lahat G, Halperin D, Fabian I, et al.
Immunomodulatory Effects of Ciprofloxacin in TNBS-Induced Colitis in
Mice. Inflamm Bowel Dis 2007; 13:557-565; ten Hove T, van den Blink B,
Pronk I, et al. Dichotomal role of inhibition of p38 MAPK with SB 203580
in experimental colitis. Gut 2002; 50:507-512; and Bouma G, Strober W.
The Immunological and Genetic Basis of Inflammatory Bowel Disease. Nature
Review Immunology 2003; 3: 521-533). The bowel in the animal model of
enteritis has swelled and the feces therein show a mushy or watery
appearance instead of a normal semi-solid type appearance. In one
embodiment, the size of the swelled bowel is obviously reduced and the
feces become soft blobs after an administration of the Bupleurum extract.
The result reveals that the extract of Bupleurum has effects on
inhibition and alleviation of IBD. In one embodiment, the level of
TNF-α, IL-6, G-CSF and IL1-β in a bowel infusion of the IBD is
significantly decreased, suggesting that these factors correlate to the
IBD.

[0039] According to the disclosure, the extract of Bupleurum is useful to
ameliorate or cure IBD in a subject, including Crohn's disease, or
enteritis or a bowel disease with an inflammatory symptom.

[0041] The subject is administrated the extract of Bupleurum in an
effective amount. The effective amount is not specifically limited, and
can be adjusted by a person skilled in the art based on a subject's age,
physical condition or inflammatory levels. The effective amount for
administration is about 50˜1,000 mg/kg or about 30˜500 mg/kg
based on the weight of the subject.

[0042] The pharmaceutical composition according to the invention may
further comprise a pharmaceutically acceptable carrier and/or additive
added in an appropriate ratio based on the dosage form, storage type or
administration route.

[0043] The administration route in the invention comprises intravenous,
intramuscular, subcutaneous or oral routes. Oral administration is
preferable. The pharmaceutical composition of the invention can be
administrated in a multiple dose regime within a period of time. The
administrative regime can be estimated and identified according to
routine pharmaceutical practices.

Example 1

Extraction of Bupleurum

[0044] The extraction of the Bupleurum species is based on U.S. patent
application Ser. No. 12/829,357 (Jul. 1, 2010) with modification.
Specifically, the roots of Bupleurum krylovianum, Bupleurum chinense,
Bupleurum commelynoideum, Bupleurum scorzonerifolium, Bupleurum
triradiatum, Bupleurum falcatum and Bupleurum kaoi were separately
grinded into powdered form.

[0045] 0.5 g of each the powdered form were added to 25 ml of an ethanol
aqueous solution in a concentration of 25%, 50%, 75% and 95% by volume,
respectively. Each of the solution was vibrated at room temperature
overnight. After being dried and concentrated, extracts of Bupleurum were
obtained, respectively.

[0051] Specifically, BABL/cAnNCrlBltw mice, purchased from BioLASCO Taiwan
(Bltw) were grouped into four groups having 6 mice to a group. In three
of the groups, each mouse was injected with 1.75 mg of trinitrobenzene
sulfonic acid (TNBS) (Sigma) at a 4 cm depth of the bowel from the anus.
After 48 hours, the injected location was induced to form enteritis. Mice
in another group were injected with a 50% ethanol aqueous solution
without TNBS at a 4 cm depth of the bowel from the anus as a negative
control (A). The location injected ethanol aqueous solution without TNBS
was not induced to form enteritis. The test article or vehicle control
was administrated at 4 hours and 24 hours after the TNBS induction, and
the animal were sacrificed after 48 hours of TNBS induction.

[0052] In one of the enteritis-induced groups, a 6 cm length cut was made
from the anus of the mice of the enteritis-induced bowel as a vehicle
treatment (B). The cut bowel was immerged in phosphate buffer saline
(PBS) for 2 hours.

[0053] In one of the enteritis-induced group, the mice were fed 100 mg/kg
of Bupleurum krylovianum extract, extracted by the extraction of the 50%
ethanol aqueous solution in Example 1. Then, a 6 cm length cut was made
from the anus of the mice of the enteritis-induced bowel as a test group
(C). The cut bowel was immerged in phosphate buffer saline (PBS) for 2
hours.

[0054] In another one of the enteritis-induced group, the mice were fed
200 mg/kg of 5-aminosalicyclic acid (5-ASA). Then, a 6 cm length cut was
made from the anus of the mice of the enteritis-induced bowel as a
positive control (D). The cut bowel was immerged in phosphate buffer
saline (PBS) for 2 hours.

[0055] The bowel infusion of the groups (A), (B), (C) and (D) were
respectively quantified and analyzed, for the amounts of TNF-α,
IL-6, G-CSF and IL1-β by an ELISA (R&D Systems®). The results
are shown in FIGS. 1˜4, respectively.

[0057] The bowels from the negative control (A), vehicle treatment (B) and
positive control (D) (200 mg/kg 5-ASAl) in Example 2 were also observed
and the swelling and the type of feces were recorded, as shown in FIGS.
5, 6 and 15, respectively.

[0058] The enteritis model in the vehicle treatment without the
administration of the Bupleurum extract showed bowel swelling and mushy
and watery feces (FIG. 6). However, in the enteritis model with the
administration of the Bupleurum extract, the bowel swelling was reduced
and soft blobs and semi-solid feces were formed (FIGS. 7˜14). The
result showed an improvement of enteritis. Meanwhile, the bowel swelling
was not alleviated or eliminated in the positive control (5-ASA); noting
that 5-ASA is currently used as the therapeutic drug for treating IBD.

[0059] While the disclosure has been described by way of example and in
terms of the embodiments, it is to be understood that the disclosure is
not limited to the disclosed embodiments. To the contrary, it is intended
to cover various modifications and similar arrangements (as would be
apparent to those skilled in the art). Therefore, the scope of the
appended claims should be accorded the broadest interpretation so as to
encompass all such modifications and similar arrangements.